Method for inducing efficient differentiation of embryonic stem cells into megakaryocytes

Abstract

The present application provides a method for inducing differentiation of embryonic stem cells into megakaryocytes. The provided method comprises the step of inducing differentiation of engineered embryonic stem cells into megakaryocytes; the engineered embryonic stem cells express a reduced amount of SHMT2 protein compared to unengineered embryonic stem cells. Megakaryocytes and platelets can be efficiently prepared via the method.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for inducing embryonic stem cells to differentiate into megakaryocytes efficiently. Background Technology

[0002] Platelets, a key component of human blood, play a vital role in blood clotting and hemostasis. In a healthy body, platelet levels remain relatively constant. When platelet levels fall below the lower limit of normal, the risk of bleeding increases significantly. Clinically, platelet transfusion is widely used to treat thrombocytopenia caused by various factors. However, due to the limited number of donors, the difficulty of platelet collection, the stringent storage conditions, and the short shelf life of platelets, the supply of platelets has long been insufficient to meet current clinical needs.

[0003] Platelets originate from megakaryocytes in organs such as bone marrow and lungs. Although megakaryocytes are extremely rare in the human body (accounting for only 0.05% of nucleated cells in bone marrow), their powerful platelet-producing capacity (each megakaryocyte can produce more than 500 platelets on average) effectively maintains platelet counts at a physiological homeostasis. Therefore, exogenous megakaryocyte transfusion, as an alternative therapy to platelet component transfusion, shows promising application prospects in treating thrombocytopenia caused by various factors.

[0004] To address the bottleneck of scarce in vivo megakaryocyte sources and difficulties in isolation, we utilized human pluripotent stem cells (HPS cells) as a new seed source for in vitro megakaryocyte preparation, employing an in vitro stem cell differentiation strategy. HPS cells possess high self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into megakaryocytes and mature platelets under specific in vitro induction conditions. Researchers have been tirelessly exploring ways to establish efficient and stable megakaryocyte differentiation systems. These explorations primarily focus on creating a differentiation microenvironment that mimics megakaryocyte development, and have gradually developed optimization strategies such as stromal cell co-culture, screening and adding cytokines and small molecule compounds, and constructing three-dimensional differentiation structures.

[0005] However, due to insufficient understanding of the development and differentiation mechanisms of megakaryocytes, the preparation of megakaryocytes derived from human pluripotent stem cells currently suffers from problems such as low induction efficiency and weak in vitro platelet production capacity of differentiated cells, which have become obstacles to the clinical application of stem cell-derived megakaryocytes. Summary of the Invention

[0006] One objective of this invention is to address one of the aforementioned problems to a certain extent. This invention provides a method for inducing embryonic stem cells to differentiate efficiently into megakaryocytes. This method can be used to efficiently prepare megakaryocytes.

[0007] This invention was completed based on the following research conducted by the inventors:

[0008] The inventors discovered during their research that mitochondrial-mediated cellular metabolism plays a crucial role in the regulation of stem cell proliferation and differentiation. For example, when hematopoietic stem cells maintain their resting state, their energy supply mainly relies on glycolysis. However, when hematopoietic stem cells differentiate into downstream blood cells, the cells rapidly switch to an energy supply state dominated by oxidative phosphorylation. This energy metabolic transition is accompanied by an increase and activation of intracellular mitochondria. The research also revealed that a gene called serine hydroxymethyltransferase 2 (SHMT2) may play an important role in hematopoietic development. SHMT2 is an enzyme found only in mitochondria, playing a vital role in cellular serine metabolism, single-carbon metabolism, and tRNA synthesis. SHMT2-mediated serine catabolism is essential for the correct translation, initiation, and maintenance of methionine-tRNAs. Furthermore, as an enzyme that plays a crucial role in cellular respiration and mitochondrial metabolism, SHMT2 likely also plays an important role in the determination of pluripotent stem cell hematopoietic fate. Therefore, the inventors knocked out the SHMT2 gene from human embryonic stem cell lines and designed a culture system to directionally induce the differentiation of human embryonic stem cells into megakaryocytes. They discovered that SHMT2 knockout embryonic stem cells possess stronger megakaryocyte differentiation potential, and the differentiation efficiency into the megakaryocyte lineage is significantly improved. The technical solution provided by this invention offers a new technical approach for the efficient preparation of megakaryocytes and platelets derived from human embryonic stem cells.

[0009] Therefore, a first aspect of the present invention provides a method for inducing embryonic stem cells to differentiate into megakaryocytes, comprising: inducing modified embryonic stem cells to differentiate into megakaryocytes; wherein the modified embryonic stem cells express a lower amount of SHMT2 protein compared to unmodified embryonic stem cells.

[0010] According to embodiments of the present invention, the modified embryonic stem cells are obtained by at least one of the following methods:

[0011] (1) This includes silencing or reducing the expression of the SHMT2 gene in embryonic stem cells;

[0012] (2) This includes using SHMT2 inhibitors to silence or reduce the expression of the SHMT2 gene in embryonic stem cells.

[0013] A second aspect of the present invention provides a method for inducing embryonic stem cells to differentiate into megakaryocytes, comprising:

[0014] (1) The modified embryonic stem cells were subjected to a first induction treatment using a first culture medium to obtain a first induction product, wherein the first culture medium contained bone morphogenetic protein-4 (BMP-4), activin A, CHIR-99021 and basic fibroblast growth factor (bFGF).

[0015] (2) The first induction product is subjected to a second induction treatment using a second culture medium to obtain a second induction product, wherein the second culture medium contains bFGF, SB431542 and vascular endothelial growth factor (VEGF);

[0016] (3) The second induction product is subjected to a third induction treatment using a third culture medium to obtain a third induction product, wherein the third culture medium contains recombinant human stem cell factor (SCF), thrombopoietin (TPO), vascular endothelial growth factor, interleukin-3 (IL-3) and Fms-like tyrosine kinase 3 ligand (Flt3-L).

[0017] According to embodiments of the present invention, the method for inducing embryonic stem cells to differentiate into megakaryocytes described above further includes the following technical features:

[0018] According to an embodiment of the present invention, the first culture medium in step (1) comprises:

[0019] IMDM basal medium (Iscove's Modified Dulbecco's Medium)

[0020] Ham's F-12 Nutrient Mixture (F-12 basal medium)

[0021] Bovine serum albumin,

[0022] cholesterol,

[0023] Linolenic acid

[0024] Linoleic acid

[0025] L-Ascorbic acid 2-phosphate,

[0026] α-Monothioglycerol (α-MTG),

[0027] Glutamine,

[0028] PFHM-II medium (Protein-Free Hybridoma Medium II)

[0029] Insulin-Transferrin-Selenium

[0030] Penicillin-Streptomycin

[0031] BMP-4,

[0032] Activin A,

[0033] CHIR-99021,

[0034] bFGF.

[0035] According to an embodiment of the present invention, the duration of the first induction treatment in step (1) is 24 to 48 hours.

[0036] According to an embodiment of the present invention, the second culture medium in step (2) comprises:

[0037] IMDM basal medium,

[0038] F-12 basal culture medium,

[0039] Bovine serum albumin,

[0040] cholesterol,

[0041] Alpha-linolenic acid,

[0042] Linoleic acid,

[0043] L-Ascorbic acid-2-phosphate,

[0044] α-MTG,

[0045] Glutamine,

[0046] PFHM-II medium,

[0047] Insulin-transferrin-selenium

[0048] Penicillin-Streptomycin

[0049] bFGF,

[0050] SB431542,

[0051] VEGF.

[0052] According to an embodiment of the present invention, the second induction treatment in step (2) takes 3 to 5 days.

[0053] According to an embodiment of the present invention, the third culture medium in step (3) comprises:

[0054] IMDM basal medium,

[0055] F-12 basal culture medium,

[0056] Bovine serum albumin,

[0057] SyntheChol NS0 Supplement

[0058] Linolenic acid,

[0059] Linoleic acid,

[0060] L-Ascorbic acid 2-phosphate,

[0061] α-MTG,

[0062] Glutamax,

[0063] Protein-Free Hybridoma Medium II,

[0064] Ihsulin-Transferrin-Selenium,

[0065] Penicillin-Streptomycin

[0066] SCF,

[0067] TPO,

[0068] VEGF,

[0069] IL-3,

[0070] Flt3-L.

[0071] According to an embodiment of the present invention, the third induction treatment in step (2) takes 5 to 10 days.

[0072] According to an embodiment of the present invention, the modified embryonic stem cells are obtained by gene knockout.

[0073] According to an embodiment of the present invention, the modified embryonic stem cells are modified mammalian embryonic stem cells, such as modified human embryonic stem cells.

[0074] A third aspect of the invention provides a megakaryocyte obtained by the method described in the first or second aspect of the invention. Attached Figure Description

[0075] Figure 1 A schematic diagram of a LentiCRISPR v2 recombinant plasmid containing an sgRNA sequence targeting the SHMT2 gene, provided according to an embodiment of the present invention.

[0076] Figure 2 The figure shows the comparison results of the transcriptional and protein expression levels of the SHMT2 gene in SHMT2-KO-H9 cells and WT-H9 cells provided by the embodiments of the present invention.

[0077] Figure 3 This is a diagram showing the comparison of transcriptional and protein expression of pluripotency-related genes between WT-H9 cells and SHMT2-KO-H9 cells according to an embodiment of the present invention.

[0078] Figure 4 The results of flow cytometry detection of pluripotency surface markers in WT-H9 cells and SHMT2-KO-H9 cells are provided according to embodiments of the present invention.

[0079] Figure 5 This is a schematic diagram of the hESC-to-megakaryocyte induction differentiation system provided by an embodiment of the present invention.

[0080] Figure 6 The figure shows the effect of SHMT2 knockout on the generation of megakaryocytes on days 10 and 14 of H9 cells induced to differentiate into hematopoietic cells, according to an embodiment of the present invention. Detailed Implementation

[0081] The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention. In this document, when the content is expressed as a percentage (%), unless otherwise specified, the percentage refers to a volume percentage. For example, when referring to the amount of each component contained in a first, second, or third culture medium, % represents the volume percentage of each component in the total volume of the culture medium.

[0082] This invention provides a method for inducing embryonic stem cells to differentiate into megakaryocytes, comprising:

[0083] A method for inducing embryonic stem cells to differentiate into megakaryocytes includes:

[0084] The steps for inducing modified embryonic stem cells to differentiate into megakaryocytes;

[0085] Compared to unmodified embryonic stem cells, the modified embryonic stem cells expressed a lower amount of SHMT2 protein.

[0086] In at least some embodiments, the modified embryonic stem cells are obtained by the following methods:

[0087] (1) This includes silencing or reducing the expression of the SHMT2 gene in embryonic stem cells;

[0088] (2) This includes using SHMT2 inhibitors to silence or reduce the expression of the SHMT2 gene in embryonic stem cells.

[0089] In at least some embodiments, the method for inducing embryonic stem cells to differentiate into megakaryocytes includes:

[0090] (1) The modified embryonic stem cells are subjected to a first induction treatment using a first culture medium to induce their differentiation into mesodermal cells and obtain a first induction product, wherein the first culture medium contains BMP-4, Activin A, CHIR-99021 and bFGF; by adding these cytokines to the culture medium, embryonic stem cells can be induced to differentiate into mesodermal cells.

[0091] (2) The first induction product is subjected to a second induction treatment using a second culture medium to induce its differentiation into hematopoietic endothelium and obtain a second induction product, wherein the second culture medium contains bFGF, SB431542 and VEGF; by adding these cytokines to the culture medium, the differentiation of mesodermal cells into hematopoietic endothelium can be promoted.

[0092] (3) The second induction product is subjected to a third induction treatment using a third culture medium to induce its differentiation into megakaryocytes in order to obtain the third induction product. The third culture medium contains SCF, TPO, VEGF, IL-3, and Flt3-L. By adding these cytokines to the culture medium, the differentiation of hematopoietic endothelial cells into megakaryocytes can be promoted.

[0093] In at least some embodiments, the first culture medium in step (1) comprises: IMDM basal medium (Iscove's Modified Dulbecco's Medium), F-12 basal medium (Ham's F-12 Nutrient Mixture), bovine serum albumin, cholesterol, linolenic acid, linoleic acid, L-ascorbic acid 2-phosphate, α-monothioglycerol (α-MTG), glutamine, PFHM-II medium (Protein-Free Hybridoma Medium II), insulin-transferrin-selenium, penicillin-streptomycin, BMP-4, Activin A, CHIR-99021, and bFGF. The application of this culture medium composition can induce embryonic stem cells to differentiate into mesodermal cells.

[0094] The cholesterol mentioned can be SyntheChol NS0 Supplement. The PFHM-II medium mentioned is a non-animal-derived, serum-free, and protein-free medium. The glutamine mentioned can be GlutaMAX. TM Additives, which have better stability, can improve cell health.

[0095] In at least some preferred embodiments, the first culture medium comprises: IMDM basal medium, F-12 basal medium, 2–5 μg / mL bovine serum albumin, 0.1–0.5% cholesterol, 0.1 μg / mL linolenic acid, 0.1 μg / mL linoleic acid, 0.05 mg / mL L-ascorbic acid-2-phosphate, 0.003%–0.005% α-thioglycerol, 0.5%–2% glutamine, 3%–8% PFHM-II medium, 0.5%–2% insulin-transferrin-selenium, 0.5%–2% penicillin-streptomycin, 10–30 ng / mL BMP-4, 10–30 ng / mL activin, 1–3 μmol / L CHIR-99021, and 10–30 ng / mL bFGF.

[0096] In at least some embodiments, the duration of the first induction treatment in step (1) is 24 to 48 hours.

[0097] According to an embodiment of the present invention, the second culture medium in step (2) comprises: IMDM basal medium, F-12 basal medium, bovine serum albumin, cholesterol, linolenic acid, linoleic acid, L-ascorbic acid-2-phosphate, α-thioglycerol, glutamine, PFHM-II medium, insulin-transferrin-selenium, penicillin-streptomycin, bFGF, SB431542, and VEGF. If the composition of the culture medium changes, the induction effect will be worse. In at least some preferred embodiments, the second culture medium in step (2) comprises: IMDM basal medium, F-12 basal medium, 2-5 μg / mL bovine serum albumin, 0.1%-0.5% cholesterol, 0.05-0.3 μg / mL linolenic acid, 0.05-0.3 μg / mL linoleic acid, 0.02-0.1 mg / mL L-ascorbic acid-2-phosphate, 0.002%-0.005% α-thioglycerol, 0.5%-3% glutamine, 3%-8% PFHM-II medium, 0.5%-3% insulin-transferrin-selenium, 0.5%-3% penicillin-streptomycin, 10-30 ng / mL bFGF, 1-5 μmol / L SB431542, and 30-80 ng / mL VEGF.

[0098] In at least some embodiments, the second induction treatment in step (2) takes 3 to 5 days.

[0099] In at least some embodiments, the third culture medium in step (3) includes: IMDM basal medium, F-12 basal medium, bovine serum albumin, cholesterol, linolenic acid, linoleic acid, L-ascorbic acid-2-phosphate, α-MTG, glutamine, PFHM-II medium, insulin-transferrin-selenium, penicillin-streptomycin, SCF, TPO, VEGF, IL-3, and Flt3-L. In at least some embodiments, the third culture medium comprises: IMDM basal medium, F-12 basal medium, 1–5 μg / mL bovine serum albumin, 0.1%–0.5% cholesterol, 0.05–0.5 μg / mL linolenic acid, 0.05–0.5 μg / mL linoleic acid, 0.01–0.3 mg / mL L-ascorbic acid-2-phosphate, 0.002–0.005% α-MTG, 0.5–5% glutamine, 3%–8% PFHM-II medium, 0.5–5% insulin-transferrin-selenium, 0.5–5% penicillin-streptomycin, 30–80 ng / mL SCF, 10–30 ng / mL TPO, 10–30 ng / mL VEGF, 10–30 ng / mL IL-3, and 10–30 ng / mL Flt3-L. If the composition of the culture medium changes, the induction effect will be worse.

[0100] In at least some embodiments, the third induction treatment in step (2) lasts for 5 to 10 days.

[0101] In at least some embodiments, the modified embryonic stem cells are obtained through gene knockout. For example, the SHMT2 gene can be knocked out from human embryonic stem cell lines using CRISPR-Cas9 technology. An inducible SHMT2 knockdown hESC cell line is constructed, and hematopoietic differentiation is induced in the inducible SHMT2 knockdown hESC cell line using an embryomimetabolite-induced differentiation method. SHMT2 is knocked down in the induced cells at specific differentiation stages to promote megakaryocyte production. In at least some embodiments, an SHMT2 inhibitor is added to the hESC cell line at different stages of embryomimetabolite-induced differentiation to promote megakaryocyte production. In at least some embodiments, an SHMT2 knockout hESC cell line is constructed, and hematopoietic differentiation is induced in the SHMT2 knockout hESC cell line using an embryomimetabolite-induced differentiation method to promote megakaryocyte production.

[0102] In at least some embodiments, the modified embryonic stem cells are modified mammalian embryonic stem cells, such as modified human embryonic stem cells.

[0103] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0104] Example 1

[0105] Example 1 describes the construction of a SHMT2 gene knockout human embryonic stem cell line using CRISP technology, including the following:

[0106] To construct an SHMT2 gene knockout (SHMT2-KO) H9 cell line, CRISPR / Cas9 technology was used, employing a LentiCRISPR v2 recombinant plasmid (e.g., one containing an sgRNA sequence targeting the SHMT2 gene) that already contained the SHMT2 gene-targeting sgRNA sequence. Figure 1 As shown in the image, the plasmid was transfected into H9 cells using electroporation. It targets exon 4 of the SHMT2 gene.

[0107] The nucleic acid sequence is as follows:

[0108] SHMT2: 5'-CAACCTCACGACCGGATCAT-3' (SEQ ID NO: 1).

[0109] (1) H9 cells were cultured in a CO2 incubator using mTeSR stem cell culture medium and the medium was changed daily.

[0110] (2) Passage H9 cells when they are in the logarithmic growth phase and have good cell morphology (cell confluence is about 70%), and pre-coat them with embryonic stem cell matrix gel for more than one hour. Preheat the culture medium to 37°C.

[0111] (3) Prepare electroporation buffer (82 μL Nucleofector Solution + 18 μL Supplement) in advance and add 5 μg SHMT2-KO recombinant plasmid. Digest H9 cells using TrypLE Select Enzyme and count cells using a cell viability analyzer, taking 8 × 10⁶ cells each time. 5 Each cell undergoes electroporation.

[0112] (4) Centrifuge the cell suspension at 115×g for 5 min, discard the supernatant, resuspend the cells in the previously prepared electroporation buffer, and transfer them into an electroporation cuvette. (Vessels)

[0113] (5) Place the electroporation cup on the holder of the electroporation instrument (Lonza), set the electroporation program (select stem cell electroporation program, CB-150), and complete the electroporation process.

[0114] (6) Resuspend the cells in preheated culture medium (using the pipette provided in the kit, gently blow two to three times to mix the cells, avoiding repeated aspiration of the sample). Seed the cells into six-well plates coated with matrix gel and incubate in a CO2 incubator.

[0115] (7) Since the LentiCRISPR v2 recombinant plasmid carries the puromycin resistance gene sequence, cells carrying the target plasmid can be screened by treating electrotransfected cells with puromycin.

[0116] After electroporation, H9 cells were continuously screened for three weeks using mTeSR medium containing 2.5 μg / mL puromycin. Clones forming single cells were then selected and screened again using mTeSR medium containing 2.5 μg / mL puromycin to identify puromycin-resistant cells. The transcriptional and protein expression levels of the SHMT2 gene in these selected cells were detected using qPCR and Western blot.

[0117] Figure 2 This figure shows a comparison of the transcriptional and protein expression levels of the SHMT2 gene between SHMT2-KO-H9 and WT-H9 cells. Figure 2Figure A shows the qPCR results of SHMT2 gene expression in SHMT2-KO-H9 and WT-H9 cells, with TBP as an internal control (n=3). Statistical analysis of the qPCR results showed that SHMT2 gene mRNA expression was undetectable in SHMT2-KO-H9 cells compared to WT-H9 cells.

[0118] Figure 2 Figure B shows the Western blot results of SHMT2-KO-H9 cells and WT-H9 cells, used to detect SHMT2 protein expression. The results show that, compared to the WT group, the SHMT2-KO group cells did not show a band at the location corresponding to the SHMT2 protein.

[0119] Figure 2 Image C shows the immunofluorescence staining results of SHMT2-KO-H9 and WT-H9 cells for SHMT2 and TOMM20 proteins, bar = 10 μm. Immunofluorescence staining was performed on cells from the WT and SHMT2-KO groups grown on confocal dishes using SHMT2, TOMM20 (mitochondrial marker protein) antibodies and DAPI. Laser scanning confocal microscopy results showed that the expression regions of SHMT2 protein (red fluorescence) and TOMM20 protein (green fluorescence) in the WT group highly overlapped, surrounding the DAPI-stained nucleus (blue fluorescence). In contrast, the SHMT2-KO group showed almost no red fluorescence, and the fluorescence intensity of TOMM20 protein (green fluorescence) and DAPI (blue fluorescence) in the SHMT2-KO group was similar to that in the WT group. This experiment demonstrates the successful construction of an SHMT2 knockout H9 cell line. The SHMT2-completely knocked-out H9 cell line was named SHMT2-KO-H9 cells.

[0120] Example 2

[0121] Example 2: Pluripotency identification of SHMT2 knockout H9 cell line.

[0122] (1) Detection of the expression level of pluripotency-related genes in SHMT2 knockout H9 cells.

[0123] Figure 3 The results show the comparison of the transcriptional and protein expression of pluripotency-related genes between WT-H9 and SHMT2-KO-H9 cells. Figure 3Table A shows the statistical results of qPCR detection of OCT4, SOX2, and NANOG gene expression in WT-H9 and SHMT2-KO-H9 cells, with TBP as an internal control (*P<0.05, n=3). The expression of pluripotency marker genes, including OCT4, SOX2, and NANOG, in SHMT2-KO-H9 cells was detected using qPCR. The results showed no significant difference in the transcriptional levels of the three pluripotency marker genes, OCT4, SOX2, and NANOG, between SHMT2-KO-H9 and WT-H9 cells.

[0124] Figure 3 Image B shows immunofluorescence staining images of OCT4, SOX2, and NANOG proteins in WT-H9 and SHMT2-KO-H9 cells (bar = 100 μm). Immunofluorescence staining was used to detect the expression of three pluripotency marker proteins (OCT4, SOX2, and NANOG) in WT-H9 and SHMT2-KO-H9 cells (green fluorescence). SHMT2 protein (red fluorescence) and cell nuclei (DAPI, blue fluorescence) were also labeled. Laser scanning confocal microscopy showed that H9 cells after SHMT2 knockout still expressed the three pluripotency marker proteins OCT4, SOX2, and NANOG. The fluorescence intensity of these three pluripotency marker proteins and DAPI after immunofluorescence staining in the SHMT2-KO group was similar to that in the WT group, and the expression locations of these three proteins highly overlapped with the locations of DAPI-labeled cell nuclei.

[0125] (2) Flow cytometry detection of pluripotency-related surface markers in SHMT2 knockout H9 cells

[0126] The expression levels of stem cell pluripotency surface markers are also important indicators of cell stemness. Flow cytometry was used to detect the expression of cell surface stemness marker proteins. Figure 4 The results of flow cytometry detection of pluripotency surface markers in WT-H9 and SHMT2-KO-H9 cells were obtained.

[0127] Figure 4 Image A shows the flow cytometry results of SSEA4 antibody and TRA-1-60 antibody labeling in WT-H9 and SHMT2-KO-H9 cells. The results indicate that SSEA4 in SHMT2-KO-H9 cells... + The cell percentage was (97.28±1.77)%, TRA-1-60 + The cell percentage was (95.95±0.68)%; SSEA4 in WT-H9 cells + The cell percentage was (96.44±4.2)%, TRA-1-60 + The cell percentage was (90.19±3.56)%.

[0128] Figure 4 Figure B shows the statistical analysis results of the flow assay for pluripotency surface markers in WT-H9 and SHMT2-KO-H9 cells (n=3). The statistical analysis results showed that SSEA4 in SHMT2-KO-H9 cells... + With TRA-1-60 + The cell percentage was not significantly different from that of WT-H9.

[0129] Example 3

[0130] Example 3 established a system for inducing hESC differentiation into megakaryocytes, and its overall schematic diagram is shown below. Figure 5 As shown.

[0131] (1) The first stage of hESC-induced differentiation:

[0132] Prepare the culture medium for the first stage of hESC induction of hematopoietic cell differentiation according to the table below:

[0133]

[0134]

[0135] b. WT-H9 and SHMT2-KO-H9 cells in the logarithmic growth phase were digested using TrypLE Select Enzyme, and the cells were resuspended in an appropriate amount of PBS. Cell counts were performed using a cell viability analyzer.

[0136] c. Using mTeSR medium containing 10 μmol / L Y-276322HCl, the two groups of cells were cultured at 2 × 10⁻⁶ cells / mL. 5 The cells were seeded at a density of / well in a low-adsorption six-well plate and cultured for 24 h to allow them to aggregate and form embryoid bodies (EBs) spheres. The cell spheres were then precipitated by centrifugation at 1000 r / min for 5 min.

[0137] d. Replace the culture medium with the medium for the first stage of hESC induction of hematopoietic cell differentiation, change the medium every other day, and culture for 48 hours. (2) Second stage of hESC induction of differentiation:

[0138] Prepare the culture medium for the second stage of hESC induction of hematopoietic cell differentiation according to the table below:

[0139]

[0140]

[0141] b. After completing the first stage of induction, the cells were centrifuged at 1000 r / min for 5 min to precipitate them. The culture medium in the induction system was replaced with the hESC induction medium for the second stage of differentiation into hematopoietic cells. The cells were cultured continuously for 4 days, and the cell medium was changed every other day.

[0142] (3) The third stage of hESC-induced differentiation:

[0143] a. Prepare the culture medium for the third stage of hESC induction of hematopoietic cell differentiation according to the table below:

[0144]

[0145] After completing the second stage of induction, the cells were centrifuged at 1000 r / min for 5 min to precipitate them. The culture medium in the induction system was then replaced with the third stage hESC induction and differentiation medium for hematopoietic cells. The cells were cultured continuously for 8 days, with the cell medium being changed every other day.

[0146] Example 4

[0147] Example 4 describes the flow cytometry detection of megakaryocyte production during the induction process.

[0148] Figure 6 The figure shows the effect of SHMT2 knockout on megakaryocyte production on days 10 and 14 of H9 cell induction into hematopoietic cells. The results showed that H9 cells with SHMT2 knockout produced a higher yield of megakaryocytes than H9 cells without SHMT2 knockout.

[0149] Figure 6 Figure A shows the results of multicolor flow cytometry analysis of the expression of CD34 and CD41, and CD41 and CD42b in two groups of cells.

[0150] Figure 6 B represents the CD34 produced by the WT group and the SHMT2-KO group on days 10 and 14 of induction of differentiation into hematopoietic cells. + CD41 + and CD41 + CD42b + Statistical analysis results of the percentage of cells in the total number of cells (*P<0.05, n=3).

[0151] Figure 6 C represents the CD34 produced on days 10 and 14 of induction of differentiation into hematopoietic cells in the WT group and SHMT2-KO group. + CD41 + and CD41 + CD42b + Statistical analysis results of cell count (*P<0.05, n=3).

[0152] Experimental results showed that after 14 days of induction, SHMT2 knockout H9 cells produced CD34 on days 10 and 14. + CD41 + and CD41 + CD42b + The proportion and number of cells were significantly higher than those in the control group.

[0153] In this document, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0154] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," and "some implementations" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0155] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for inducing embryonic stem cells to differentiate into megakaryocytes, comprising: The steps for inducing modified embryonic stem cells to differentiate into megakaryocytes; The modified embryonic stem cells are SHMT2 knockout embryonic stem cells; These include: (1) using a first culture medium to induce the modified embryonic stem cells to obtain a first induction product, wherein the first culture medium contains BMP-4, activin, CHIR-99021 and bFGF; (2) The first induction product is subjected to a second induction treatment using a second culture medium to obtain a second induction product, wherein the second culture medium contains bFGF, SB431542 and VEGF; (3) The second induction product is subjected to a third induction treatment using a third culture medium to obtain a third induction product, wherein the third culture medium contains SCF, TPO, VEGF, IL-3 and Flt3-L.

2. The method according to claim 1, characterized in that, The first culture medium in step (1) includes: IMDM basal medium, F-12 basal culture medium, Bovine serum albumin, cholesterol, Alpha-linolenic acid, Linoleic acid, L-Ascorbic acid-2-phosphate, α-Thioglycerol, Glutamine, PFHM-II medium, Insulin-transferrin-selenium Penicillin-Streptomycin BMP-4, Activator CHIR-99021, bFGF.

3. The method according to claim 2, characterized in that, The first culture medium comprises: IMDM basal medium, F-12 basal culture medium, 2~5 μg / mL bovine serum albumin, 0.1-0.5% cholesterol, 0.1 μg / mL linolenic acid, 0.1 μg / mL linoleic acid, 0.05 mg / mL L-ascorbic acid-2-phosphate, 0.003%~0.005% α-thioglycerol, 0.5%~2% glutamine, 3%~8% PFHM-II medium, 0.5%~2% insulin-transferrin-selenium, 0.5%~2% penicillin-streptomycin, 10~30 ng / mL of BMP-4, Activin at concentrations of 10-30 ng / mL CHIR-99021 at concentrations of 1–3 μmol / L 10~30 ng / mL of bFGF.

4. The method according to claim 1, characterized in that, The first induction treatment in step (1) lasts for 24 to 48 hours.

5. The method according to claim 1, characterized in that, The second culture medium in step (2) includes: IMDM basal medium, F-12 basal culture medium, Bovine serum albumin, cholesterol, Alpha-linolenic acid, Linoleic acid, L-Ascorbic acid-2-phosphate, α-Thioglycerol, Glutamine, PFHM-II medium, Insulin-transferrin-selenium Penicillin-Streptomycin bFGF, SB431542, VEGF.

6. The method according to claim 5, characterized in that, The second culture medium includes: IMDM basal medium, F-12 basal culture medium, 2~5 μg / mL bovine serum albumin, 0.1%~0.5% cholesterol, 0.05~0.3μg / mL linolenic acid, 0.05~0.3μg / mL linoleic acid, 0.02~0.1 mg / mL of L-ascorbic acid-2-phosphate, 0.002%~0.005% α-thioglycerol, 0.5%~3% glutamine, 3%~8% PFHM-II medium, 0.5%~3% Insulin-Transferrin-Selenium 0.5%~3% penicillin-streptomycin 10~30 ng / mL of bFGF, SB431542 at concentrations of 1~5 μmol / L VEGF at concentrations of 30-100 ng / mL.

7. The method according to claim 1, characterized in that, Step (2) The second induction treatment lasts for 3 to 5 days.

8. The method according to claim 1, characterized in that, The third culture medium mentioned in step (3) includes: IMDM basal medium, F-12 basal culture medium, Bovine serum albumin, cholesterol, Alpha-linolenic acid, Linoleic acid, L-Ascorbic acid-2-phosphate, α-MTG, Glutamine, PFHM-II medium, Insulin-transferrin-selenium Penicillin-Streptomycin SCF, TPO, VEGF, IL-3, Flt3-L.

9. The method according to claim 8, characterized in that, The third culture medium includes: IMDM basal medium, F-12 basal culture medium, 1~5 μg / mL bovine serum albumin, 0.1%~0.5% cholesterol, 0.05~0.5 μg / mL linolenic acid, 0.05~0.5 μg / mL linoleic acid, 0.01~0.3 mg / mL L-ascorbic acid-2-phosphate, 0.002~0.005% α-MTG, 0.5-5% glutamine, 3%~8% PFHM-II medium, 0.5-5% insulin-transferrin-selenium 0.5-5% penicillin-streptomycin SCF 30~80 ng / mL 10~30 ng / mL of TPO, VEGF at concentrations of 10-30 ng / mL 10~30 ng / mL of IL-3, 10~30 ng / mL of Flt3-L.

10. The method according to claim 1, characterized in that, The third induction treatment in step (2) lasts for 5 to 10 days.

11. The method according to any one of claims 1-10, characterized in that, The modified embryonic stem cells are modified mammalian embryonic stem cells.

12. The method according to claim 11, characterized in that, The modified embryonic stem cells are modified human embryonic stem cells.

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